The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Synthetic oligonucleotides tethered to various ligands have been used as research tools in molecular biology [for reviews, see e.g. Iyer, R. P., Roland, A., Zhou, W., and Ghosh, K. Curr. Opin. Mol. Ther., 1999, 1, 344; Uhlman, E. and Peyman. A. Chem Rev, 1990, 90, 543; English. U. and Gauss, D. H. Angew. Chem. Int. Ed. Engl. 1991, 30, 613. Beaugace and Iyer, Tetrahedron, 1993, 49, 6123.; Wojczewski, C., Stolze, K. and Engels, J. W. Synlett, 1999, 1667]. They have been applied to genetic analysis, and to elucidate mechanism of gene function. Oligonucleotides carrying reporter groups have had widespread use for automated DNA sequencing, hybridization affinity chromatography and fluorescence microscopy, Oligonucleotide-biotin conjugates axe widely used as hybridization probes. Antisense oligonucleotides covalently linked to intercalators, chain cleaving or alkylating agents have been shown to be efficient as gene expression regulators. The sequence specific artificial nucleases, when targeted against mRNA, may find applications even as chemotherapeutics.
The labels can be attached to the target oligonucleotides or polynucleotides either chemically or enzymatically. The chemical approach includes often preparation of modified building blocks, and their subsequent insertion into synthetic oligonucletides during oligonucleotide synthesis. Alternatively, natural DNA can be transformed, for example by bisulfite catalyzed transamination of cytosine residues [Biochemistry, 1980, 19, 1774] followed by labeling of the amino functions with appropriate label molecules, The enzymatic approach, in turn, consists of preparation of nucleoside triphosphates derivatized with appropriate tether molecules and their incorporation into RNA or DNA structure by a polymerase reaction.
For several applications, such as for DNA hybridization assays, it is desirable to introduce more than one reporter group to the oligonucleotide structure. This can be performed by three alternative methods: (i) by coupling several base- or carbohydrate-tethered nucleosidic building blocks or nucleoside triphosphates to the growing oligonucleotide chain, (ii) by functionalization of the internucleosidic phosphodiester linkages, or (iii) by using several multifunctional non-nucleosidic building blocks during oligonucleotide chain assembly. All of these methods have their own drawbacks. Since the double helix formation of DNA is based on hydrogen bonding between the complementary base residues, tethers attached to the base moieties often weaken these interactions. This problem is easily overcome by using the tethered nucleosides at the 3′- or 5′-terminus of the coding sequence, or by using labels linked to C5 of pyrimidine residues. Introduction of tethers to the phosphate backbone gives rise to new chiral centers and makes the purification of these analogues difficult. Introduction of the tether arm to the carbohydrate moiety, in turn, often decreases the coupling efficiency of the phosphoramidite due to steric hindrance.
Introduction of linker arms to the nucleobase is most commonly performed by allowing a nucleoside with a good leaving group (N-tosyl, N-benzoyl, halogen, triazole, thiol) at C4 of pyrimidines or C2, C8 or C6 of purines to react with the appropriate nucleophilic linker molecule (e.g. an alkane-α,ω-diamine). Since normally an excess of linker molecule and rather vigorous reaction conditions has to be used laborious purification procedures cannot be avoided. The basic reaction conditions needed gives additional requirements to the other protecting groups in the target molecule. These problems may be overcome by attachment of the linker molecules to C5 of pyrimidine bases by a palladium catalyzed coupling reaction between 5-halogeno pyrimidine nucleoside and an alkynyl- or allyl linker. Recently, attachment of linker arm to the N3 of 3′, 5′-O-protected thymidine [J. Org. Chem., 1999, 64, 5083; Nucleosides, Nucleotides, 1999, 18, 1339] and 2′-deoxy-5′-O-(4,4′-dimethoxytrityl)uridine [Org. Lett. 2001, 3, 2473] based on Mitsunobu reaction have been reported. Since the coupling reaction is performed under mild conditions, a wide range of tethers can be introduced.
The majority of methods described in literature involve attachment of functional groups in the oligonucleotide structure during chain assembly. Hence, introduction of the label molecules has to be performed in solution. In the labeling reaction the amino or mercapto groups of oligonucletides are allowed to react in solution with isothiocyanato, haloacetyl or 2,4,6-triazinyl derivatives of label molecules. Carboxylic acid groups, in turn, can be labeled with amino tethered labels with the aid of water-soluble carbodiimide. Since in all the cases the labeling reaction is performed in aqueous solution with an excess of labeling reactants, laborious purification procedures cannot be avoided. Especially when attachment of several labels is required the isolation and characterization of the desired conjugate is extremely difficult, and often practically impossible. Hence, several attempts to incorporate label molecules or their appropriately protected precursor to oligonucleotide structure during chain assembly have been done [U.S. Pat. No. 4,948,882, U.S. Pat. No. 5,583,236]. The fluorescent label monomers for solid phase chemistry synthesized are most commonly organic dyes (e.g. fluorescein, rhodamine, dansyl, dabsyl, pyrene, Alexa, Cy, TAMRA), several of these blocks are even commercially available. However, such labels and labeled biomolecules suffer from many commonly known drawbacks such as Raman scattering, other fluorescent impurities, low water solubility, concentration quenching etc. In specific binding assays, generally very low concentrations of analytes to be measured are present. Thus multilabeling of oligonucleotides with organic fluorophores may not enhance detection sensitivity the extent needed in many applications. For this type of applications lanthanide(III) chelates are labels of choice since they do not suffer from this phenomenon. In DNA hybridization assays, time-resolved luminescence spectroscopy using lanthanide chelates is well known [Hemmila et al. Bioanalytical Applications of Labelling Technologies, Wallac Oy, 1994]. Therefore, a number of attempts have been made to develop new highly luminescent chelate labels suitable for time-resolved fluorometric applications. These include e.g. stabile chelates composed of derivatives of pyridines [U.S. Pat. No. 4,920,195, U.S. Pat. No. 4,801,722, U.S. Pat. No. 4,761,481, PCT/FI91/00373, U.S. Pat. No. 4,459,186, EP Appl.0770610, Remuinan et al, J. Chem. Soc. Perkin Trans 2, 1993, 1099], bipyridines [U.S. Pat. No. 5,216,134], terpyridines [U.S. Pat. No. 4,859,777, U.S. Pat. No. 5,202,423, U.S. Pat. No. 5,324,825] or various phenolic compounds [U.S. Pat. No. 4,670,572, U.S. Pat. No. 4,794,191, Ital Pat. 42508 A789] as energy mediating groups and polycarboxylic acids as chelating parts. In addition, various dicarboxylate derivatives [U.S. Pat. No. 5,032,677, U.S. Pat. No. 5,055,578, U.S. Pat. No. 4,772,563] macrocyclic cryptates [U.S. Pat. No. 4,927,923, WO 93/5049, EP-A493745] and macrocyclic Schiff bases [EP-A-369-000] have been published. Also a method for the labeling of a biospecific binding reactant such as hapten, a peptide, a receptor ligand, a drug or PNA oligomer with luminescent labels by using solid-phase synthesis has been published [U.S. Pat. No. 6,080,839, EP 067205A1]. Also oligonucleotide labeling reagents have been synthesized and used in multilabeling of oligonucleotides [Nucleic Acids Res., 22, 1994, Org. Lett., 2001, 3, 2473].
For several applications, such as for those involving an antisense approach, enhanced stability of oligonucleotides towards nucleases is desired. Most commonly this has been achieved by modifying the phosphodiester backbone (mono- and dithioates, phosphoramidites) or carbohydrate moiety.